Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction, scarring, and hypertrophy leading to heart failure. The myocardium adapts to this ischemic injury by inducing scar formation which is crucial to stabilize the ischemic area and prevent rupture; however, cardiac fibrosis often develops in these patients over time. Therefore, post-MI adaptation of the heart is crucial for its long term functio and clinical management of cardiac fibrosis post-MI presents a major challenge. The differentiation of CFs to myofibroblasts is a critical first step following myocardial infarction ad disproportionate production and prolonged survival of myofibroblasts can generate excessive ECM protein deposition leading to pathological fibrosis. Although TGFb1 and mechanical stress (generated by ECM stiffness) are recognized as major mediators of myofibroblast differentiation, the molecular signals that coordinate these soluble and mechanical signals are still elusive. The long-term objective of this proposal is to understand the mechanotransduction mechanisms of cardiac fibroblast differentiation to myofibroblasts. Our preliminary results demonstrate that the mechanosensitve ion channel TRPV4 (Transient Receptor Potential Vanilloid 4) is required for TGF-b1- induced differentiation of CF into myoFibs. Importantly, we have found that TGF-b1-induced myofibroblast differentiation required a high stiffness matrix, a response that was attenuated by TRPV4 blockade. TGF-b1 treatment enhanced TRPV4 expression, membrane translocation (via p38 MAPK) and activity in CFs. Further, we found that TRPV4 activates a mechanosensitive transcription factor, MRTF-A (myocardin related transcription factor-A), via Rho-dependent mechanism. Importantly, we found that when subjected to MI, cardiac function was preserved and fibrosis was reduced in the TRPV4KO mice compared to wild type mice. Finally, injection of Rho activator, CNF1 in to the hearts of TRPV4KO mice caused cardiac fibrosis in post-MI hearts confirming the role of this mechanotransduction pathway in the mediation of cardiac fibrosis. Our working hypothesis is that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts through the integration of chemical (TGF-b1) and mechanical signaling. We will test this hypothesis in the following specific aims 1) Define the molecular mechanisms by which TGF-b1 regulates TRPV4 expression, trafficking, and activity in cardiac fibroblasts 2) Interrogate the mechanotransduction mechanisms by which TRPV4 mediates cardiac fibroblast differentiation and 3) Determine a causative role of TRPV4 in cardiac fibrosis following myocardial infarction. TRPV4 channels have not been studied as potential therapeutic targets for myocardial infarction. Our proposed studies will establish TRPV4 as an integrator of mechanical and chemical signals required for myocardial adaptation to ischemia and may open entirely new avenues for development therapeutics for myocardial ischemia and pathological cardiac fibrosis.